Enzyme-linked competitive binding assays are well recognized as alternatives to radioimmunoassays for the sensitive and selective determination of a variety of biologically important analytes (ligands). An inherent drawback of these assays is associated with the fact that multi-subsituted enzyme-ligand conjugates are typically being used. This increases the affinity of the binder for the conjugate (i.e., the binder favors the conjugate over the analyte) and results in assays with poor (not optimum) detection limits. In this study, we propose to develop and evaluate novel enzyme-linked competitive binding assays for biomolecules by taking advantage of the selective interaction between biological binders (antibodies, binding proteins, receptors, etc.) and ligands, as well as the sensitivity associated with enzyme-amplified detection. Instead of multi-substituted, mono-substituted enzyme-ligand conjugates (i.e., conjugates where one molecule of ligand is attached per enzyme molecule) are proposed. Theoretical models predict that the use of such conjugates could improve the overall dose-response characteristics of the enzyme- linked assays. Three different approaches to the synthesis of mono-substituted conjugates will be undertaken. First, enzymes possessing covalently attached prosthetic groups can be also viewed as mono-substituted conjugates and will be used in their natural state for the assay of their corresponding prosthetic groups (e.g., biotin, lipoic acid, etc.). Second, ligand-substituted enzymes will be prepared by incubating apoenzymes with ligand- prosthetic group conjugates. Therefore, the resulting holoenzymes will be, essentially, mono-substituted with the ligand. Finally, a substrate-or coenzyme-directed photoaffinity labeling procedure will be used to attach a ligand to the vicinity of the active site of enzymes. We intend to study the effect that specific biological binders have on the catalytic activity of these enzyme-ligand conjugates. Specifically, systems in which formation of a complex between the conjugate and its corresponding biological binder will cause inhibition of the enzymatic activity of the conjugate will be optimized in order to develop simple homogeneous assays for ligands in physiological samples. In cases where no substantial inhibition of the enzymatic activity is observed, the development of solid phase heterogeneous type assays will be investigated. In both cases, basic fundamental studies will be undertaken to investigate the nature of the observed assay characteristics and to improve the sensitivity and detection limits of the technique.